Maternal alcohol abuse during pregnancy can result in Fetal Alcohol Spectrum Disorders (FASD), a lifelong disability characterized by a range of neuroanatomical and neurobehavioral deficits. Current estimates of FASD prevalence is about 2-5% among young school children in the United States. Therefore, early detection of alcohol use among pregnant women is critical to prevent FASD. Maternal self-reporting is largely unreliable and the utility of current biomarkers is also limited during pregnancy. Among these markers, glycosylated variants of the protein transferrin (carbohydrate deficient transferrin, CDT), is reported to be the most diagnostically specific biomarker for chronic alcohol abuse. However, the mean sensitivity is about ~65.4% with a wide range depending upon the analytical method employed. Moreover, utilizing the conventional electrophoretic, chromatographic, and immunometric methods, it has so far been difficult to characterize the composition and abundance of all glycoforms. This is critical because alcohol produces complex effects on the glycans including loss of sialic acids, partial loss of sugars, and/or complete loss of oligosaccharide chains. In a widely utilized FASD rat model, we have for the first time utilized state of the art mass spectrometry and obtained preliminary data characterizing the sequences and abundances of maternal plasma transferrin N- linked oligosaccharides preserving the location of the N-glycosite. Based on our novel data, we propose the following Specific Aim: Develop and validate a novel and sensitive high throughput platform utilizing a traditional biomarker (variants of transferrin) for maternal alcohol consumption screening in a FASD rat model. Two sub aims are proposed to characterize and validate the protein's post-translational glycosylation signature abundance profile in the maternal plasma and relating it to the dose and duration of alcohol exposure. We will utilize a pregnant rat model exposed to graded alcohol doses administered for different durations of exposure during gestation. Plasma protein (transferrin) identification, sequence mapping, glycopeptide identification with diagnostic fragmentation spectra, and High Resolution/Accurate Mass quantitation will be performed. Validation studies will include N-linked Glycosite confirmation and lectin-based probing. Based on our preliminary data, we expect the proposed platform to provide a highly sensitive protein post-translational modification signature profile that will greatly enhance the window of detection and also clearly distinguish between the alcohol doses. At the end, we plan to correlate the glycosylation signature profile with FASD neurobehavioral measures. We also believe that these novel methodologies can be in the future extended to pregnant women and form the basis for a high throughput screening tool that will help establish a standardized diagnostic criteria with a real clinical impact not only in diagnosis but also in treatment and prevention.